Fig 2: The formation of ISL-1/Set7/9/PDX-1 complex was regulated by IGF-1 in an age-related manner. (A) NIT-1 cells were treated with mouse recombinant IGF-1 protein. The H3k4me3 of the CyclinD1 promoter was verified by ChIP assay. (B) The whole cell lysates of NIT-1 cells treated with IGF-1 were used for Western blotting. (C) The enrichment of ISL-1, Set7/9 and PDX-1 at the CyclinD1 promoter was monitored by ChIP assay in NIT-1 cells treated with IGF-1. (D, E) Co-IP assays were performed to measure the ISL-1/Set7/9/PDX-1 complex in NIT-1 cells treated with IGF-1. ISL-1 and GAPDH served as a loading control and negative controls, respectively. (F) Rat islet lysates were subjected to ChIP assay using anti-ISL-1, PDX-1, Set7/9 antibodies or normal IgG. (G) Rat islet lysates were immunoprecipitated by anti-ISL-1 antibody. Western blotting was employed to detect the endogenous interaction using anti-PDX-1 or anti-Set7/9 antibodies. (H) The formation of the ISL-1/PDX-1/Set7/9 complex was detected by Co-IP assay in the islets of “young” or “old” rats. The data in (A), (C) and (F) represent 3 independent experiments, each performed in triplicate. *p < 0.05, **p < 0.01, #p < 0.05, ##p < 0.01 vs. the control, NS, non-significance.

Fig 3: ISL-1 recruited Set7/9 to modulate histone modification of the CyclinD1 promoter. (A) H3k4me3 was detected by ChIP assay in HIT-T15 cells at indicated time-points after serum starvation for 48 h. The H3K4me3 level at 0 h serum starvation served as a control. (B, C) pcDNA3.1-ISL-1 (ISL-1), pcDNA3.1-vector (control), ISL-1 siRNA or Non-silencer negative control siRNA were transfected into HIT-T15 cells. ChIP assays were employed to detect H3k4me1 or H3k4me3 after 48 h. (D) The luciferase activity of the CylinD1 promoter was measured in 293A cells transfected with the indicated plasmid combination. The cells transfected with pcDNA3 were used as a negative control. (E) NIT-1 cells was transfected with pCMV-Set7/9 (pCMV-vector served as a control) or Set7/9 siRNA (Non-silencer served as a control), and Western blotting analysis was performed using an antibody against H3k4me3. Histone H3 was used as a loading control. (F, G) NIT-1 cells were transfected with different combinations of plasmids for 48 h, and H3k4me3 or H3k4me1 of the CyclinD1 promoter were measured by ChIP assay. The cells transfected with pcDNA3 or Non-silencer were used as negative controls. *p < 0.05, **p < 0.01, vs. pcDNA3; #p < 0.05, ##p < 0.01 vs. Non-silencer, NS: non-significance.

Fig 4: L3MBTL3 interacts with the methylated K142 to regulate the stability of the methylated DNMT1 protein. a The interaction between L3MBTL3 and DNMT1 is dependent on SET7 and K142. The Flag-WT-DNMT1 and its K142A mutant and SET7 constructs were co-transfected into HCT116 cells. The interactions of DNMT1 and the mutant with L3MBTL3 were analyzed by anti-L3MBTL3 immunoprecipitation and western blotting as indicated. b SET7 stimulated the methylation of K142 and interaction between DNMT1 and L3MBTL3. SET7 expression constructs were transfected into HCT116 cells. The interactions between DNMT1 and L3MBTL3 and the methylated DNMT1 were analyzed by immunoprecipitation and western blotting with anti-methylated K142 and other indicated antibodies. c L3MBTL3 preferentially binds to the monomethylated K142 peptide. Sulfolink-immobilized DNMT1 peptides containing the monomethylated (Kme1) or cognate unmethylated K142 (Kme0) were incubated with 250 µg of HCT116 extracts for 4 h. L3MBTL3 binding to the peptide resins was analyzed by anti-L3MBTL3 Western blotting and quantified with independent Student’s t-test. ***P < 0.001. d L3MBTL3 directly interacts with the monomethylated K142 peptide. The K142me0 and Kme1 peptide resins were pre-incubated with 5 µg of GST for 2 h to reduce the background. The resins were then incubated with 1.5 µg of GST-L3MBTL3 or its D381N mutant for 4 h at 4 °C as indicated. The washed resins were blotted with anti-L3MBTL3 antibodies. e Mutation of the MBT domain in L3MBTL3 reduces its binding to DNMT1 in vivo. HA-tagged L3MBTL3 (WT) and the D381N mutant expression constructs were transfected into HCT116 cells for 42 h and then treated with MG-132 for additional 6 h. The interaction between DNMT1 and the wild-type or mutant HA-L3MBTL3 proteins were examined by reciprocal immunoprecipiation and western blotting with anti-DNMT1 or anti-HA antibodies. Student’s t-test was calculated for significant differences. ***P < 0.001. All quantifications were represented by bar graph with mean and S.D. for error bars from three replicates and all experiments were repeated three independent times with the same conclusion

Fig 5: L3MBTL3 interacts with CRL4DCAF5 and recruits DCAF5 onto the methylated DNMT1 protein. a Downregulation of DCAF5 or L3MBTL3 increases the K142-methylated DNMT1. HCT116 cells were transfected with 50 nM of control, DCAF5 and L3MBTL3 siRNAs. DNMT1 was immunoprecipitated by anti-DNMT1 antibody and western blotted by anti-methylated K142 or anti-DNMT1 antibodies. Student’s t-test was caculated for significant differences. **P < 0.01 and ***P < 0.001. b L3MBTL3 and DCAF5 interact. Endogenous DCAF5 and L3MBTL3 proteins were immunoprecipitated from HCT116 cells and western blotted with indicated antibodies. c The interaction between DCAF5 and DNMT1 is abolished by K142 mutation. Flag-WT-DNMT1 and its K142A mutant constructs were transfected into HCT116 cells and the interactions between DCAF5 and Flag-DNMT1 or the K142A mutant were analyzed by anti-DCAF5 immunoprecipitation and western blotting with anti-Flag and DCAF5 antibodies. d Downregulation of L3MBTL3 abolished the interaction between DNMT1 and DCAF5. Endogenous DNMT1 and DCAF5 proteins were immunoprecipitated by anti-DNMT1 antibodies from control and L3MBTL3 siRNA-mediated knockdown cells (42 h of siRNA and MG132 for additional 6 h). The immunoprecipitated proteins were blotted with anti-DCAF5 and DNMT1 antibodies. e L3MBTL3 is required for preferential binding of DCAF5 to the monomethylated K142 peptide. HCT116 extracts expressing a stable Flag-DCAF5 or Flag-DCAF5 and L3MBTL3 were prepared. 250 µg extract proteins were incubated with the monomethylated K142 or the cognate unmethylated peptide resins as in Fig. 5c. The binding of Flag-DCAF5 to the peptide resins with or without L3MBTL3 were examined by anti-Flag and L3MBTL3 antibodies. Student’s t-test was used for significant differences. *P < 0.05. f L3MBTL3 and CRL4DCAF5 target DNMT1 for polyubiquitination in vivo. Flag-WT DNMT1 and Flag-K142 A mutant expressing constructs were co-transfected into 293 cells together with vectors expressing HA-tagged ubiquitin (HA-Ub), SET7, CUL4A, CUL4B, and DDB1, in the presence or absence of L3MBTL3 and DCAF5 expressing constructs as indicated. Proteins were immunoprecipitated with anti-HA antibodies and western blotted with the anti-Flag-DNMT1 antibodies as indicated. Quantifications for all experiments were represented by bar graph with mean and S.D. for error bars from three replicates and Student’s t-test was used for significant differences, as described in Fig. 2a